This renewal continues our previous efforts to better define various elements within the coronary microcirculation that regulate blood flow, and also furthers the elucidation of the signal transduction pathway for flow-induced dilation, which we believe is a response that facilitates communication of metabolic signals throughout the coronary circulation. The first objective is to elucidate a critical factor that is one of the causal links between coronary blood flow and myocardial oxygen consumption. Accordingly, aim 1 test the hypothesis that hydrogen peroxide (H2O2) links coronary blood flow to myocardial metabolism. Our second objective is to elucidate the basis and effects of cardiac myocyte-produced metabolites on endothelial function. This aim will test the hypothesis that during increased metabolism, cardiac myocytes produce a factor that induces endothelial cell depolarization and decreases NO production. Our third objective is to define the signaling pathway by flow induced EDHF-mediated vasodilation. This last aim will test the hypothesis that the signal transduction pathways for flow-induced EDHF- and NO-mediated dilation are different. Our experimental approach incorporates a variety of techniques including Western analyses of activated proteins to understand signal transduction pathways, patch clamping to identify ionic mechanisms associated with FID, isolated microvessels to elucidate FID transduction pathways, isolated microvessels and myocytes to identify metabolic hyperemic mechanisms, fluorescence microscopy to assess production of reactive oxygen species (ROS), viral transfection of the myocardium, and in vivo studies establishing the link between myocardial metabolism and coronary blood flow. The elucidation of these aims will facilitate our understanding of the causal basis of metabolic hyperemia, the relationships between metabolic and endothelium-dependent dilation, and the mechanotransduction of shear stress leading to EDHF-mediated dilation. This information will greatly aid our understanding of the regulation of coronary blood flow.